The invention first relates to a device comprising a substrate, sensor layer capable of binding magnesium ions and a scavenging layer that preferentially binds to calcium ions in the presence of both magnesium ions and calcium ions. The present invention also relates to a method of determining the concentration of magnesium ions in a sample wherein the luminoionophore is contacted with magnesium ion in a sample, wherein the intensity of at least one fluorescence emission changes and the concentration of magnesium ion is calculated based on the change in the intensity of the emission. The present invention also relates to novel luminoionophores, comprising a luminophoric moiety and an ionophoric moiety, capable of binding magnesium.
The accurate measurement of physiologic cations, such as sodium, potassium, lithium, calcium, and magnesium, is essential in clinical diagnosis. Traditionally, these ions were determined in plasma or serum using ion-selective electrodes (ISE), which are very cumbersome to use and costly to maintain. Serious drawbacks of electrochemical measuring arrangements are the requirement of a reference element, sensitivity towards electrical potentials and electromagnetic interference.
An alternative enzymatic method is based on the activation of β-Galactosidase by cations (Berry et al., Clin. Chem., 34/11, 1988 2295-2298). However, the high cost and poor stability of the enzyme preclude its extensive application in clinical laboratories. Therefore, the development of practical and inexpensive colorimetric reagents for the clinical determination of these ions in biological fluids remains an important area of research.
U.S. Pat. No. 4,367,072 describes a process for the determination of metal ions using simple crown ethers as ion-binding units. However, the binding lacks sufficient specificity to be useful for many practical applications, such as clinical applications, in which the indicator has to discriminate between ions with very similar properties, e.g., sodium versus potassium or magnesium versus calcium.
U.S. Pat. Nos. 6,211,359; 5,952,491; and 6,171,866 (each of which is hereby individually incorporated by reference in its entirety) report ionophores for potassium, sodium, and calcium, respectively. These ionophores have π-electron conjugated nitrogen and are coupled to a fluorophore or luminophore to make fluorophore-ionophore or luminophore-ionophore sensors where the respective ions are detected by measuring fluorescence or luminescence emission. All three ionophores have been shown to be very selective in determination of potassium, sodium, and calcium in whole blood, respectively (see He et. al. Anal. Chem. Vol. 75, 2003, 449-555; and J. Am. Chem. Soc. vol. 125, 2003, 1468-1469), thus showing that the ionophores are effective at physiological pH. However, these publications do not provide for an ionophore that selectively binds magnesium.
The invention relates to determination of ions by the luminescence method based on the reversible binding of cations to a cation-selective ionophore and the so-called “PET effect” (photoinduced electron transfer) between the ionophoric and a luminophoric moiety. Determination of other ions by similar methods is described in U.S. Pat. Nos. 6,211,359; 6,171,866; and 5,952,491, which are each hereby incorporated by reference in their entirety. The cation-selective ionophore may in some instances be selective for more than one cation, but one or more cations may be excluded from binding with the ionophoric moiety by providing an additional selective ionophore, which is selective for the ion to be excluded, in a manner so that the ions must contact the selective filtering ionophore prior to contacting the ionophoric and a luminophoric moiety that shows PET effect.
The so-called “PET effect” denotes the transfer, induced by photons, of electrons from the ionophoric moiety to the luminophoric moiety, which leads to a decrease in the (relative) luminescence intensity and the luminescence decay time of the luminophore. Absorption and emission wavelengths, however, remain basically unaffected in the process (J. R. Lakowicz in “Topics in Fluorescence Spectroscopy”, Volume 4: Probe Design and Chemical Sensing; Plenum Press, New York & London (1994)).
By the binding of ions to the ionophore the PET effect is partially or completely inhibited, which results in an increase in the relative luminescence intensity and an increase in the luminescence decay time of the luminophoric moiety. Hence, one can deduce the concentration or the activity of a desired ion by measuring the luminescence properties, e.g., relative luminescence intensity and/or luminescence decay time. Activities can be related to concentrations via known Debye-Huckel formulae.
From U.S. Pat. No. 5,516,911, fluorescent indicators based on fluorinated BAPTA derivatives are known. These indicators generally have Kd values in the millimolar range. These fluorescent indicators, however, suffer from a relatively complicated synthesis of the fluorinated BAPTA derivatives.
Moreover, the known ionophores based on BAPTA or on derivatives thereof in an aqueous environment and at normal ambient temperatures are previously shown to exhibit some chemical instability (see, e.g., U.S. Pat. No. 4,603,209, column 26, lines 40-46). This is particularly disadvantageous in determination procedures using optical sensors in measuring situations requiring a high shelf life (durability) of the sensor or where, for monitoring purposes, one sensor is to be used for measuring over prolonged time periods. Often these compounds display smaller Kd values for cations other than magnesium, such as calcium.
The present invention avoids and overcomes the disadvantages and problems in the prior art. The present invention has as its object to provide luminoionophores and devices for the optical determination of magnesium ions, whose ionophores are more easily synthesizable, and can be covalently bound to suitable luminophores when in electronically decoupled condition. Furthermore, the ionophores need not show high selectivity for magnesium ions because a blocking layer comprising an ionophore which preferentially binds to an alternate competing cation, such as calcium, can be installed between the luminoionophore and the solution containing cation.
In addition, the luminoionophores may be bound to a hydrophilic polymer material by means of a chemical group in order to use them in optical sensors.
The luminoionophore should not exhibit inherent pH dependence in the expected pH range of the sample and should be excitable by light of commercially available LEDs, for example at wavelengths >420 nm. These luminoionophores should, in addition, be chemically stable in an aqueous environment even at high ambient temperatures and over prolonged time periods.